Method for preventing or treating nosocomial pneumonia

ABSTRACT

A method for preventing or treating nosocomial diseases, e.g., diseases caused by  Pseudomonas aeruginosa , is provided. The method includes administering to a susceptible human a specified dose of a bispecific antibody that that specifically binds  Pseudomonas aeruginosa  Psl and PcrV.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 15/779,189, now U.S. Pat. No. 11,066,461, which is a 35 U.S.C. § 371National Phase Application of International Application No.PCT/US2016/063865, filed Nov. 28, 2016, which claims the benefit of U.S.Provisional Application No. 62/260,935, filed Nov. 30, 2015, the contentof each of which is incorporated herein by reference in its entirety.

REFERENCE TO A SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA EFS-WEB

The content of the electronically submitted sequence listing (Name:2943_1160002_Seqlisting_ST25.txt; Size: 22,646 bytes; and Date ofCreation: Jun. 21, 2021) is herein incorporated by reference in itsentirety.

BACKGROUND

Pseudomonas aeruginosa (P. aeruginosa) is an important nosocomialpathogen, and pneumonia is one of its most concerning manifestations.Mechanical ventilation is the most important risk factor for nosocomialpneumonia caused by P. aeruginosa, with cumulative risk increasing withthe duration of ventilation (Zahar, J R, et al. Crit Care Med. 2009September; 37(9):2545-51). In one study, P. aeruginosa accounted for 13%of cases of microbiologically confirmed pneumonia acquired in theintensive care unit (ICU) by patients who were not mechanicallyventilated, and 24% of cases in those who were (Esperatti et al, Am JRespir Care Med. 2010 Dec. 15; 182(12):1533-9). In a review of globalepidemiology data, P. aeruginosa caused approximately 27% ofventilator-associated pneumonia (VAP; Jones, Clin Infect Dis. 2010 Aug.1; 51 Suppl 1:S81-7). Respiratory tract colonisation with P. aeruginosais another important risk factor (Rehm and Kollef, Poster #367. 43rdCritical Care Congress, Society of Critical Care Medicine (SCCM); 9-13Jan. 2014, San Francisco Calif., USA). Despite the existence ofantibiotics, pneumonia, particularly VAP, due to P. aeruginosa remainsassociated with significant mortality and morbidity, increased ICU andhospital length of stay, and substantial economic burden.

Patients in the ICU are at risk for developing serious pneumoniainfection and mechanical ventilation increases the risk for pneumonia inthe ICU. P. aeruginosa is a leading cause of ICU pneumonia thatcontributes significantly to increased hospital stays (55.4 days v. 7.2days), ICU stays (14.8 days v. 1.1 days), and greater need formechanical ventilation (62.3% v. 7.4%), as well as patient mortality(20.2% v. 3.1% of). (Kyaw M H, et al. J Infect Dis. (2005)192(3):377-386). Additionally, multi-drug resistance has complicated themanagement of P. aeruginosa infection. With limited antimicrobialtherapeutic options, consideration of new approaches, such asimmunoprophylaxis for the prevention of P. aeruginosa would address animportant unmet need.

MEDI3902 is a bivalent, bispecific human immunoglobulin G1 (IgG1) kappamonoclonal antibody (mAb) that selectively binds to both the PcrVprotein and Psl exopolysaccharide on the surface of P. aeruginosa.Pharmacology studies have demonstrated that MEDI3902 is capable ofmediating three distinct mechanisms of action: anticytotoxicity,opsonophagocytosis and killing, and inhibition of P. aeruginosaattachment to cells. Binding to PcrV on intact P. aeruginosa preventstype 3 secretion (T3S) injectisome-mediated cytotoxicity and damage tohost cells. Binding to Psl mediates opsonophagocytic killing (OPK) of P.aeruginosa by host effector cells and inhibits P. aeruginosa attachmentto host epithelial cells (DiGiandomenico et al, J Exp Med. 2012 Jul.2:209(7):1273-87).

MEDI3902 was highly protective in P. aeruginosa murine infection models.See, e.g., in PCT Publication No. WO 2013/070615, PCT Publication No. WO2014/074528, PCT Application No. PCT/US2015/029063, and PCT ApplicationNo. PCT/US2015/036576. Moreover, MEDI3902 provided synergisticenhancement of antibiotic therapy against P. aeruginosa pneumonia withdistinct antibiotic classes against both antibiotic-sensitive andantibiotic-resistant P. aeruginosa.

SUMMARY

This disclosure provides a method of preventing or treating nosocomialinfection in a susceptible human subject, where the method includesadministering to the subject about 500 to about 3000 mg, e.g., about500, 600, 700, 750, 1000, 1500 or about 3000 mg, of a bispecificantibody that specifically binds Pseudomonas aeruginosa Psl and PcrV(e.g., MEDI3902), and monitoring the subject for symptoms through 21days from the day of administration. According to the method, at 21 dayspost-administration the subject can be symptom-free or can display lesssevere symptoms relative to a cohort of susceptible human subjectsadministered a placebo. In certain aspects the nosocomial infection canbe pneumonia, e.g., Pseudomonas aeruginosa pneumonia, bacteremia, boneinfection, joint infection, skin infection, burn infection, woundinfection, or any combination thereof.

This disclosure further provides a method of preventing or treatingpneumonia, e.g., Pseudomonas aeruginosa pneumonia, in a susceptiblehuman subject, where the method includes administering to the subjectabout 500 to about 3000 mg, e.g., about 500, 600, 700, 750, 1000, 1500or about 3000 mg, of a bispecific antibody that specifically bindsPseudomonas aeruginosa Psl and PcrV (e.g., MEDI3902), and monitoring thesubject for pneumonia symptoms through 21 days from the day ofadministration. According to the method, at least at 21 dayspost-administration the subject can be pneumonia-free or can displayless severe symptoms relative to a cohort of susceptible human subjectsadministered a placebo.

In some embodiments, the serum target level of the bispecific antibody(e.g., MEDI3902) is at least about 1 μg/mL, at least about 2 μg/mL, atleast about 3 μg/mL, at least about 4 μg/mL, or at least about 5 μg/mL.In other embodiments, the serum target level of the bispecific antibody(e.g., MEDI3902) is at least about 1.7 μg/mL. In further embodiments,the serum target level of the bispecific antibody (e.g., MEDI3902) is atleast about 5.3 μg/mL In some embodiments, the administration produces aserum level of the bispecific antibody (e.g., MEDI3902) of at least 1μg/mL, at least about 2 μg/mL, at least about 3 μg/mL, at least about 4μg/mL, or at least about 5 μg/mL through day 21 following administrationof the bispecific antibody. In other embodiments, the administrationproduces a serum level of the bispecific antibody (e.g., MEDI3902) of atleast 1.7 μg/mL through day 21 following administration of thebispecific antibody. In further embodiments, the serum target level ofthe bispecific antibody (e.g., MEDI3902) is at least about 5.3 μg/mL

In some embodiments, the method of preventing or treating nosocomialinfection in a susceptible human subject comprises administering abispecific antibody comprising a binding domain which specifically bindsto P. aeruginosa Psl comprising a set of complementarity determiningregions (CDRs): HCDR1-Psl, HCDR2-Psl, HCDR3-Psl, LCDR1-Psl, LCDR2-Psl,and LCDR3-Psl, wherein HCDR1-Psl has the amino acid sequence of SEQ IDNO: 10, HCDR2-Psl has the amino acid sequence of SEQ ID NO: 11,HCDR3-Psl has the amino acid sequence of SEQ ID NO: 12, LCDR1-Psl hasthe amino acid sequence of SEQ ID NO: 13, LCDR2-Psl has the amino acidsequence of SEQ ID NO: 14, and LCDR3-Psl has the amino acid sequence ofSEQ ID NO: 15; and a binding domain which specifically binds to P.aeruginosa PcrV comprising a set of CDRs: HCDR1-PcrV, HCDR2-PcrV,HCDR3-PcrV, LCDR1-PcrV, LCDR2-PcrV, and LCDR3-PcrV, wherein HCDR1-PcrVhas the amino acid sequence of SEQ ID NO: 2, HCDR2-PcrV has the aminoacid sequence of SEQ ID NO: 3, HCDR3-PcrV has the amino acid sequence ofSEQ ID NO: 4, LCDR1-PcrV has the amino acid sequence of SEQ ID NO: 6,LCDR2-PcrV has the amino acid sequence of SEQ ID NO: 7, and LCDR3-PcrVhas the amino acid sequence of SEQ ID NO: 8. In certain aspects, thebispecific antibody has a heavy chain and a light chain. The heavy chainincludes the formula VH-CH1-H1-L1-S-L2-H2-CH2-CH3, where VH is ananti-P. aeruginosa PcrV heavy chain variable domain including the aminoacid sequence of SEQ ID NO: 1, CH1 is a heavy chain constant regiondomain-1, H1 is a first heavy chain hinge region fragment, L1 is a firstlinker, S is an anti-P. aeruginosa Psl ScFv molecule including the aminoacid sequence of SEQ ID NO: 9, L2 is a second linker, H2 is a secondheavy chain hinge region fragment, CH2 is a heavy chain constant regiondomain-2, and CH3 is a heavy chain constant region domain-3. The lightchain includes the formula VL-CL, where VL is an anti-P. aeruginosa PcrVlight chain variable domain including the amino acid sequence of SEQ IDNO: 5 and CL includes an antibody light chain kappa constant region oran antibody light chain lambda region. In certain aspects, CH1 caninclude the amino acid sequence of SEQ ID NO: 16. In certain aspects L1and L2 can be the same or different, and can independently include (a)[GGGGS]n, where n is 0, 1, 2, 3, 4, or 5 (SEQ ID NO: 23), (b) [GGGG]n,where n is 0, 1, 2, 3, 4, or 5 (SEQ ID NO: 24), or a combination of (a)and (b). In certain aspects, H1 can include the amino acid sequenceEPKSC (SEQ ID NO: 17). In certain aspects, H2 can include the amino acidsequence DKTHTCPPCP (SEQ ID NO: 18). In certain aspects, CH2-CH3 caninclude the amino acid sequence of SEQ ID NO: 19. In certain aspectsCH2-CH3 can include the amino acid sequence of SEQ ID NO: 20. In certainaspects, the heavy chain of the bispecific antibody can include theamino acid sequence of SEQ ID NO: 21, and the light chain includes theamino acid sequence of SEQ ID NO: 22.

According to the methods provided herein, the bispecific antibody (e.g.,MEDI3902) can be administered as a single intravenous (IV) infusion.

According to the methods provided herein, at the time of theadministration of the bispecific antibody the subject can be colonizedwith Pseudomonas aeruginosa in the respiratory tract, e.g., the lowerrespiratory tract. In certain aspects, the subject does not havepneumonia symptoms at the time of administration. In certain aspects,the subject's respiratory tract is colonized with P. aeruginosa one,two, three, or four days prior to administration of the bispecificantibody. Colonization can be measured, e.g., by detection of P.aeruginosa in a tracheal aspirate within 36 hours prior toadministration of the bispecific antibody. In certain aspects, thesubject's respiratory tract can be additionally colonized byStaphylococcus aureus at the time of administration of the bispecificantibody.

In certain aspects, the subject is about to be hospitalized, iscurrently hospitalized, e.g., in an intensive care unit (ICU), wasrecently hospitalized, is on a mechanical ventilator, e.g., is intubatedor ventilated through an endotracheal or nasotracheal tube, or anycombination thereof. According to the methods provided herein, theadministration of the bispecific antibody can reduce the risk ofpneumonia while on mechanical ventilation, or after mechanicalventilation is no longer required.

In certain aspects a microbiologic confirmation of pneumonia can includea respiratory specimen positive for P. aeruginosa by culture, a bloodculture positive for P. aeruginosa, a pleural fluid aspirate or lungtissue culture positive for P. aeruginosa, or any combination thereof.

In certain aspects, the subject has not received antibiotics consideredactive against the P. aeruginosa strain with which the subject iscolonized prior to administration of the bispecific antibody (e.g.,MEDI3902). In other aspects, the method can further includeadministering an antibiotic to the subject. In certain aspects, the P.aeruginosa strain with which the subject is colonized can sensitive tothe antibiotic, or can be resistant or partially resistant to theantibiotic. Where an antibiotic as administered it can be administeredprior to administration of the bispecific antibody, concurrently withadministration of the bispecific antibody, following administration ofthe bispecific antibody, or any combination thereof.

In certain aspects the method can further include administering anantihistamine to the subject. Where an antihistamine is administered itcan be administered prior to administration of the bispecific antibody,concurrently with administration of the bispecific antibody, followingadministration of the bispecific antibody, or any combination thereof.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIG. 1 provides a flow diagram of the Phase IIb clinical trial studyprotocol. ADA=anti-drug antibody; IV=intravenous; N=number of subjects;PK=pharmacokinetics. Efficacy will be assessed through 21 days postdose(Day 22); safety, PK and ADA will be assessed through 49 days postdose(Day 50). The sample size can be modified after approximately 50% of thesubjects are enrolled and followed through 21 days postdose based onblinded assessment of the event rate and/or attrition rate in theoverall population.

FIG. 2 Based on observed increased clearance in ICU patients formonoclonal antibodies directed at bacterial pathogens (e.g., MEDI4893),pharmacokinetics were modelled for ICU patients with mechanicalventilation following a single dose of MEDI3902. (A) Single dose of 1500mg MEDI3902 is predicted to maintain serum exposure at about 1 μg/mLfor >21 days in >90% subjects. (B) Single dose of 3000 mg MEDI3902 ispredicted to maintain serum exposure at about 1.7 μg/mL for ≥21 daysin >90% subjects.

FIG. 3 In vivo survival study of MEDI3902-treated mice in a 6206 acutepneumonia model system. Mice (n=6) were treated with an isotype controlIgG (negative control, 0.75 mg/kg) or MEDI3902 at three doses: 1 mg/kg,0.5 mg/kg, or 0.2 mg/kg. Twenty-four hours post-treatment, all mice wereinfected with ˜2×10⁵ CFU/animal of P. aeruginosa strain 6206. All micewere monitored for 168 hours. All of the control mice succumbed toinfection by approximately 120 hours post-infection. All of theMEDI3902-treated animals survived. Results are represented asKaplan-Meier survival curves.

DETAILED DESCRIPTION Abbreviations

Abbreviations used in this disclosure are listed in Table 1.

TABLE 1 Abbreviations Abbreviation or Specialized Term Definition ADAanti-drug antibody AE adverse event AESI adverse event of specialinterest APACHE-II Acute Physiology and Chronic Health Evaluation-II AUCarea under concentration time curve AUC_(∞) area under theconcentration-time curve from time zero to infinity AUC_(Day 22-Day 29)area under concentration time curve from day 22 to day 29 BALbronchoalveolar lavage C_(max) mean observed maximum concentration CPISClinical Pulmonary Infection Score EC₉₀ effective serum concentrationassociated with 90% survival FiO₂ fraction of inspired oxygen ICUintensive care unit Ig immunoglobulin IgG1 immunoglobulin G1 ILinterleukin ITT intent-to-treat IV intravenous LLOQ lower limit ofquantification mAb monoclonal antibody O₂ oxygen OPK opsonophagocytickilling P. aeruginosa Pseudomonas aeruginosa PaO₂ partial pressure ofoxygen PCR polymerase chain reaction PD pharmacodynamic PKpharmacokinetics RBC red blood cell SAE serious adverse event SOFASequential Organ Failure Assessment TEAE treatment-emergent adverseevent TESAE treatment-emergent serious adverse event VAPventilator-associated pneumonia Vd_(ss) volume of distribution at steadystate WBC white blood cell w/v weight/volume

Definitions

It is to be noted that the term “a” or “an” entity refers to one or moreof that entity; for example, “a binding molecule,” is understood torepresent one or more binding molecules. As such, the terms “a” (or“an”), “one or more,” and “at least one” can be used interchangeablyherein.

Furthermore, “and/or” where used herein is to be taken as specificdisclosure of each of the two specified features or components with orwithout the other. Thus, the term and/or” as used in a phrase such as “Aand/or B” herein is intended to include “A and B,” “A or B,” “A”(alone), and “B” (alone). Likewise, the term “and/or” as used in aphrase such as “A, B, and/or C” is intended to encompass each of thefollowing aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; Aand C; A and B; B and C; A (alone); B (alone); and C (alone).

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure is related. For example, the ConciseDictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed.,2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed.,1999, Academic Press; and the Oxford Dictionary Of Biochemistry AndMolecular Biology, Revised, 2000, Oxford University Press, provide oneof skill with a general dictionary of many of the terms used in thisdisclosure.

Units, prefixes, and symbols are denoted in their Système Internationalde Unites (SI) accepted form. Numeric ranges are inclusive of thenumbers defining the range. Unless otherwise indicated, amino acidsequences are written left to right in amino to carboxy orientation. Theheadings provided herein are not limitations of the various aspects oraspects of the disclosure, which can be had by reference to thespecification as a whole. Accordingly, the terms defined immediatelybelow are more fully defined by reference to the specification in itsentirety.

Disclosed herein are certain binding molecules, or antigen-bindingfragments, variants, or derivatives thereof. Unless specificallyreferring to full-sized antibodies such as naturally-occurringantibodies, the term “binding molecule” encompasses full-sizedantibodies as well as antigen-binding fragments, variants, analogs, orderivatives of such antibodies, e.g., naturally-occurring antibody orimmunoglobulin molecules or engineered antibody molecules or fragmentsthat bind antigen in a manner similar to antibody molecules.

As used herein, the term “binding molecule” refers in its broadest senseto a molecule that specifically binds an antigenic determinant. Asdescribed further herein, a binding molecule can comprise one or more“binding domains.” As used herein, a “binding domain” is a two- orthree-dimensional polypeptide structure that can specifically bind agiven antigenic determinant, or epitope. A non-limiting example of abinding molecule is a bispecific antibody or fragment thereof thatcomprises at least two distinct binding domains that specifically binddifferent antigenic determinants or epitopes. In certain aspects, abispecific antibody as provided herein can be said to comprise a firstbinding domain binding to a first epitope, and a second binding domainbinding to a second epitope.

The terms “antibody” and “immunoglobulin” can be used interchangeablyherein. An antibody (or a fragment, variant, or derivative thereof asdisclosed herein comprises at least the variable domain of a heavy chainand at least the variable domains of a heavy chain and a light chain.Basic immunoglobulin structures in vertebrate systems are relativelywell understood. See, e.g., Harlow et al., Antibodies: A LaboratoryManual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988).

As will be discussed in more detail below, the term “immunoglobulin”comprises various broad classes of polypeptides that can bedistinguished biochemically. Those skilled in the art will appreciatethat heavy chains are classified as gamma, mu, alpha, delta, or epsilon,(γ, μ, α, δ, ε) with some subclasses among them (e.g., γ1-γ4). It is thenature of this chain that determines the “class” of the antibody as IgG,IgM, IgA IgG, or IgE, respectively. The immunoglobulin subclasses(isotypes) e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁, etc. are wellcharacterized and are known to confer functional specialization.Modified versions of each of these classes and isotypes are readilydiscernible to the skilled artisan in view of the instant disclosureand, accordingly, are within the scope of this disclosure.

Light chains are classified as either kappa or lambda (κ, λ). Each heavychain class can be bound with either a kappa or lambda light chain. Ingeneral, the light and heavy chains are covalently bonded to each other,and the “tail” portions of the two heavy chains are bonded to each otherby covalent disulfide linkages or non-covalent linkages when theimmunoglobulins are generated either by hybridomas, B cells orgenetically engineered host cells. In the heavy chain, the amino acidsequences run from an N-terminus at the forked ends of the Yconfiguration to the C-terminus at the bottom of each chain.

Both the light and heavy chains are divided into regions of structuraland functional homology. The terms “constant” and “variable” are usedfunctionally. In this regard, it will be appreciated that the variabledomains of both the light (VL) and heavy (VH) chain portions determineantigen recognition and specificity. Conversely, the constant domains ofthe light chain (CL) and the heavy chain (CH1, CH2 or CH3) conferimportant biological properties such as secretion, transplacentalmobility, Fc receptor binding, complement binding, and the like. Byconvention the numbering of the constant region domains increases asthey become more distal from the antigen binding site or amino-terminusof the antibody. The N-terminal portion is a variable region and at theC-terminal portion is a constant region; the CH3 and CL domains actuallycomprise the carboxy-terminus of the heavy and light chain,respectively.

As indicated above, the variable region allows the binding molecule toselectively recognize and specifically bind epitopes on antigens. Thatis, the VL domain and VH domain, or subset of the complementaritydetermining regions (CDRs), of a binding molecule, e.g., an antibodycombine to form the variable region that defines a three dimensionalantigen binding site. This quaternary binding molecule structure formsthe antigen binding site present at the end of each arm of the Y. Morespecifically, the antigen binding site is defined by three CDRs on eachof the VH and VL chains.

In naturally occurring antibodies, the six “complementarity determiningregions” or “CDRs” present in each antigen binding domain are short,non-contiguous sequences of amino acids that are specifically positionedto form the antigen binding domain as the antibody assumes its threedimensional configuration in an aqueous environment. The remainder ofthe amino acids in the antigen binding domains, referred to as“framework” regions, show less inter-molecular variability. Theframework regions largely adopt a β-sheet conformation and the CDRs formloops that connect, and in some cases form part of, the β-sheetstructure. Thus, framework regions act to form a scaffold that providesfor positioning the CDRs in correct orientation by inter-chain,non-covalent interactions. The antigen binding domain formed by thepositioned CDRs defines a surface complementary to the epitope on theimmunoreactive antigen. This complementary surface promotes thenon-covalent binding of the antibody to its cognate epitope. The aminoacids comprising the CDRs and the framework regions, respectively, canbe readily identified for any given heavy or light chain variable regionby one of ordinary skill in the art, since they have been preciselydefined (see, “Sequences of Proteins of Immunological Interest,” Kabat,E., et al., U.S. Department of Health and Human Services, (1983); andChothia and Lesk, J. Mol. Biol., 196:901-917 (1987), which areincorporated herein by reference in their entireties).

Antibodies or antigen-binding fragments, variants, or derivativesthereof include, but are not limited tomonoclonal, human, humanized, orchimeric antibodies, single chain Fvs (scFv), and multispecificantibodies, e.g., bispecific antibodies. ScFv molecules are known in theart and are described, e.g., in U.S. Pat. No. 5,892,019. Immunoglobulinor antibody molecules encompassed by this disclosure can be of any type(e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3,IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule.

By “specifically binds,” it is generally meant that a binding molecule,e.g., a bispecific antibody or fragment, variant, or derivative thereofbinds to an epitope via an antigen binding domain, and that the bindingentails some complementarity between an antigen binding domain and theepitope. A binding molecule as provided herein can contain one, two,three, four, or more binding domains that can be the same or different,and that can bind to the same epitope, or to two or more differentepitopes. According to this definition, a binding molecule is said to“specifically bind” to an epitope when it binds to that epitope, via itsantigen binding domain more readily than it would bind to a random,unrelated epitope. The term “specificity” is used herein to qualify therelative affinity by which a certain binding molecule binds to a certainepitope. For example, binding molecule “A” may be deemed to have ahigher specificity for a given epitope than binding molecule “B,” orbinding molecule “A” may be said to bind to epitope “C” with a higherspecificity than it has for related epitope “D.”

Antibody fragments including single-chain antibodies can comprise thevariable region(s) alone or in combination with the entirety or aportion of the following: hinge region, CH1, CH2, and CH3 domains. Alsoincluded are antigen-binding fragments also comprising any combinationof variable region(s) with a hinge region, CH1, CH2, and CH3 domains.Antibodies, or antigen-binding fragments thereof disclosed herein can befrom any animal origin including birds and mammals. The antibodies canbe human, murine, donkey, rabbit, goat, guinea pig, camel, llama, horse,or chicken antibodies. As used herein, “human” antibodies includeantibodies having the amino acid sequence of a human immunoglobulin andinclude antibodies isolated from human immunoglobulin libraries or fromanimals transgenic for one or more human immunoglobulins and that do notexpress endogenous immunoglobulins, as described infra and, for examplein, U.S. Pat. No. 5,939,598 by Kucherlapati et al.

As used herein, the term “light chain portion” includes amino acidsequences derived from an immunoglobulin light chain. The light chainportion comprises at least one of a VL or CL domain.

The terms “multispecific antibody” or “bispecific antibody” as usedherein refer to an antibody that has binding domains specific for two ormore different antigens or epitopes within a single antibody molecule.It will be appreciated that other molecules in addition to the canonicalantibody structure can be constructed with two binding specificities. Itwill further be appreciated that antigen binding by bispecificantibodies can be simultaneous or sequential. Triomas and hybridhybridomas are two examples of cell lines that can secrete bispecificantibodies. Bispecific antibodies can also be constructed by recombinantmeans. (Ströhlein and Heiss, Future Oncol. 6:1387-94 (2010); Mabry andSnavely, IDrugs. 13:543-9 (2010)).

As used herein, the term “engineered antibody” refers to an antibody inwhich the variable domain in either the heavy and light chain or both isaltered by at least partial replacement of one or more CDRs from anantibody of known specificity and, if necessary, by partial frameworkregion replacement and sequence changing. Although the CDRs can bederived from an antibody of the same class or even subclass as theantibody from which the framework regions are derived, it is envisagedthat the CDRs will be derived from an antibody of different class andpreferably from an antibody from a different species. An engineeredantibody in which one or more “donor” CDRs from a non-human antibody ofknown specificity is grafted into a human heavy or light chain frameworkregion is referred to herein as a “humanized antibody.” It may not benecessary to replace all of the CDRs with the complete CDRs from thedonor variable region to transfer the antigen binding capacity of onevariable domain to another. Rather, it may only be necessary to transferthose residues that are necessary to maintain the activity of the targetbinding site. Given the explanations set forth in, e.g., U.S. Pat. Nos.5,585,089, 5,693,761, 5,693,762, and 6,180,370, it will be well withinthe competence of those skilled in the art, either by carrying outroutine experimentation or by trial and error testing to obtain afunctional engineered or humanized antibody.

As used herein, the terms “treat” or “treatment” refers to therapeutictreatment, wherein the object is to clear or reduce the bacterial burdenof an infectious agent in a subject that has been clinically diagnosedwith an infection, such as pneumonia, bacteremia, peritonitis, sepsis,and/or an abscess. “Treatment” can also mean prolonging survival ascompared to expected survival if not receiving treatment. Those in needof treatment include those already with the infection, condition, ordisorder as well as those prone to have the condition or disorder orthose in which the condition or disorder is to be prevented, e.g., inburn patients or immunosuppressed patients susceptible to bacterialinfection, e.g., P. aeruginosa infection.

As used herein, the terms “prevent” or “mitigate” refer to prophylacticor preventative measures, wherein the object is to prevent or slow down(lessen) an undesired physiological change, infection, or disorder.Beneficial or desired clinical results include, but are not limited to,alleviation of symptoms, diminishment of extent of disease, stabilized(i.e., not worsening) state of disease, clearance or reduction of aninfectious agent such as P. aeruginosa in a subject, a delay or slowingof disease progression, amelioration or palliation of the disease state,and remission (whether partial or total), whether detectable orundetectable.

As used herein, the terms “nosocomial disease” and “nosocomialinfection” refer to a disease or infection occurring or originating in ahospital or other healthcare facility. Nosocomial infections aretypically infections that are contracted in a hospital or otherhealthcare facility, and can be caused by infectious agents, e.g.,bacteria that are resistant to antibiotics. In certain aspects, anosocomial infection is not present or incubating prior to the subjectbeing admitted to the hospital or healthcare facility, which is acquiredor contracted after the subject's admittance to the hospital orhealthcare facility.

Similarly, an “iatrogenic disease” or “iatrogenic infection” is one thatis caused by or occurs as a result of medical care.

By “subject” or “individual” or “animal” or “patient” or “mammal,” ismeant any subject, e.g., a mammalian subject, for whom diagnosis,prognosis, or therapy is desired. Mammalian subjects include humans,domestic animals, farm animals, and zoo, sports, or pet animals such asdogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows,bears, and so on.

As used herein, phrases such as “a subject that would benefit fromadministration of an anti-Pseudomonas aeruginosa Psl and PcrV bispecificbinding molecule” and “an animal in need of treatment” includessubjects, such as mammalian subjects, that would benefit fromadministration of an anti-Pseudomonas Psl and PcrV bispecific bindingmolecule, such as a bispecific antibody. Such binding molecules can beused, e.g., for detection of Pseudomonas Psl or PcrV (e.g., for adiagnostic procedure) and/or for treatment, i.e., palliation orprevention of a disease, with anti-Pseudomonas aeruginosa Psl and PcrVbispecific binding molecules. As described in more detail herein, theanti-Pseudomonas aeruginosa Psl and PcrV bispecific binding moleculescan be used in unconjugated form or can be conjugated, e.g., to a drug,prodrug, or an isotope.

As used herein the terms “susceptible subject” or “susceptible humansubject” refer to a subject, e.g., a human patient, who is at risk,e.g., low risk, moderate risk, or high risk, of contracting a disease,e.g., a disease associated with P. aeruginosa, because of prior orcurrent injury or disease, planned, current, or prior hospitalization,e.g., in an intensive care unit, assisted breathing, e.g., viamechanical ventilation through intubation or a tracheostomy, and/orknown colonization with P. aeruginosa, e.g., in the respiratory tract,where the P. aeruginosa can be, in some instances, antibiotic resistant.In certain aspects a subject colonized with P. aeruginosa can besymptom-free, or can exhibit mild, moderate, or severe disease symptoms,e.g, pneumonia symptoms as described elsewhere herein. A prior orcurrent infection can be, e.g., a burn infection, a joint infection, awound infection, a skin infection, an intra-abdominal infection,bacteremia, peritonitis, sepsis, an abscess, a bone infection, or acombination of two or more such infections. In certain aspects thesubject can have, or be at risk of contracting acute pneumonia, burninjury, corneal infection, cystic fibrosis, or a combination thereof. Incertain aspects, a susceptible human subject can be treated with abispecific antibody as provided herein to prevent disease, e.g.,nosocomial disease, iatrogenic disease, or a disease caused by P.aeruginosa, from occurring, or to mitigate, e.g., alleviate, reduce,diminish, lessen, weaken, lighten, attenuate, palliate, or relieve,disease or infection symptoms resulting from P. aeruginosa infection, orto treat an infection resulting from P. aeruginosa, e.g., eliminating orlowering the bacterial burden of a P. aeruginosa infection. In certainaspects a susceptible human subject is a subject in need of treatment,e.g., based on risk factors (as noted above) for contracting a diseasetreatable by the methods provided herein, or because of existingsymptoms that require treatment.

Bispecific Antibodies

The methods provided by this disclosure utilize bispecific antibodies orantigen-binding fragments thereof, which specifically bind toPseudomonas aeruginosa Psl and PcrV. The bispecific antibodies orfragments thereof as disclosed herein comprise polypeptides, e.g., aminoacid sequences encoding, for example, Psl-specific and PcrV-specificantigen binding regions derived from immunoglobulin molecules. Apolypeptide or amino acid sequence “derived from” a designated proteinrefers to the origin of the polypeptide. In certain cases, thepolypeptide or amino acid sequence that is derived from a particularstarting polypeptide or amino acid sequence has an amino acid sequencethat is essentially identical to that of the starting sequence, or aportion thereof, wherein the portion consists of at least 10-20 aminoacids, at least 20-30 amino acids, at least 30-50 amino acids, or thatis otherwise identifiable to one of ordinary skill in the art as havingits origin in the starting sequence.

Exemplary bispecific antibodies for use in the methods provided hereinare disclosed, e.g., in PCT Publication No. WO 2013/070615, PCTPublication No. WO 2014/074528, PCT Application No. PCT/US2015/029063,and PCT Application No. PCT/US2015/036576, the disclosures of which areincorporated by reference herein in their entireties.

Exemplary portions of a bispecific antibody for use in the methodsprovided herein are presented in Table 2. The CDRs in the VH, VL, andscFv sequences are underlined. The linker sequences are doubleunderlined.

TABLE 2 Sequences SEQ ID NO: Description Amino Acid Sequence 1 Anti-PcrVEVQLLESGGG LVQPGGSLRL SCAASGFTFS SYAMNWVRQA PGKGLEWVS VHAITMSGITAYY TDDVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAKEEFLPGTHYYYG MDVWGQGTTV TVSS 2 HCDR1-PcrV SYAMN 3 HCDR2-PcrVAITMSGITAYYTDDVKG 4 HCDR3-PcrV EEFLPGTHYYYGMDV 5 Anti-PcrVAIQMTQSPSS LSASVGDRVT ITCRASQGIR NDLGWYQQKP GKAPKLLIYS VLASTLQSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCLQ DYNYPWTFGQ GTKVEIK 6LCDR1-PcrV RASQGIRNDLG 7 LCDR2-PcrV SASTLQS 8 LCDR3-PcrV LQDYNYPWT 9Anti-PsI QVQ LQESGPGLVK PSETLSLTCT VSGGSISPYY WTWIRQPPGK CLELIGYIHS scFvSGYTDYNPSL KSRVTISGDT SKKQFSLKLS SVTAADTAVY YCARADWDRLRALDIWGQGT MVTVSSGGGG SGGGGSGGGG SGGGGSDIQL TQSPSSLSASVGDRVTITCR ASQSIRSHLN WYQQKPGKAP KLLIYGASNL QSGVPSRFSGSGSGTDFTLT ISSLQPEDFA TYYCQQSTGA WNWFGCGTKV EIK 10 HCDR1-PsI PYYWT 11HCDR2- PsI YIHSSGYTDYNPSLKS 12 HCDR3- PsI ADWDRLRALDI 13 LCDR1- PsIRASQSIRSHLN 14 LCDR2- PsI GASNLQS 15 LCDR3- PsI QQSTGAWNW 16 CH1ASTKGP SVFPLAPSSK STSGGTAALG CLVKDYFPEP VTVSWNSGALTSGVHTFPAV LQSSGLYSLS SVVTVPSSSL GTQTYICNVN HKPSNTKVDK RV 17 H1 EPKSC 18H2 DKTHTCPPCP 19 CH2-CH3 APELLGGPSVFLFPPKPKDTLX₁IX₂RX₃PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPSLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK,wherein X₁ is M or Y, X₂ is S or T, and X₃ is T or E 20 CH2-CH3APELLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVSHEDPEVKFNWYVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGKEYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSREE MTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPV LDSDGSFFLY SKLTVDKSRWQQGNVFSCSV MHEALHNHYT QKSLSLSPGK 21 MEDI3902EVQLLESGGG LVQPGGSLRL SCAASGFTFS SYAMNWVRQA PGKGLEWVS HeavyAITMSGITAYY TDDVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAKEE ChainFLPGTHYYYG MDVWGQGTTV TVSS ASTKGP SVFPLAPSSK STSGGTAALGCLVKDYFPEP VTVSWNSGAL TSGVHTFPAV LQSSGLYSLS SVVTVPSSSLGTQTYICNVN HKPSNTKVDK RVEPKSCGGG GSGGGGS QVQ LQESGPGLVKPSETLSLTCT VSGGSISPYY WTWIRQPPGK CLELIGYIHS SGYTDYNPSLKSRVTISGDT SKKQFSLKLS SVTAADTAVY YCARADWDRL RALDIWGQGTMVTVSSGGGG SGGGGSGGGG SGGGGSDIQL TQSPSSLSAS VGDRVTITCRASQSIRSHLN WYQQKPGKAP KLLIYGASNL QSGVPSRFSG SGSGTDFTLTISSLQPEDFA TYYCQQSTGA WNWFGCGTKV EIKGGGGSGG GGSDKTHTCPPCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVSHEDPEVKFNWYVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGKEYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSREE MTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPV LDSDGSFFLY SKLTVDKSRWQQGNVFSCSV MHEALHNHYT QKSLSLSPGK 22 MEDI3902AIQMTQSPSS LSASVGDRVT ITCRASQGIR NDLGWYQQKP GKAPKLLIYS LightASTLQSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCLQ DYNYPWTFGQ ChainGTKVEIK RTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKVDNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC 

The bispecific antibody to be administered according to the methodsherein includes a binding domain which specifically binds to P.aeruginosa Psl comprising a set of complementarity determining regions(CDRs): HCDR1-Psl, HCDR2-Psl, HCDR3-Psl, LCDR1-Psl, LCDR2-Psl, andLCDR3-Psl, wherein HCDR1-Psl has the amino acid sequence of SEQ ID NO:10, HCDR2-Psl has the amino acid sequence of SEQ ID NO: 11, HCDR3-Pslhas the amino acid sequence of SEQ ID NO: 12, LCDR1-Psl has the aminoacid sequence of SEQ ID NO: 13, LCDR2-Psl has the amino acid sequence ofSEQ ID NO: 14, and LCDR3-Psl has the amino acid sequence of SEQ ID NO:15; and a binding domain which specifically binds to P. aeruginosa PcrVcomprising a set of CDRs: HCDR1-PcrV, HCDR2-PcrV, HCDR3-PcrV,LCDR1-PcrV, LCDR2-PcrV, and LCDR3-PcrV, wherein HCDR1-PcrV has the aminoacid sequence of SEQ ID NO: 2, HCDR2-PcrV has the amino acid sequence ofSEQ ID NO: 3, HCDR3-PcrV has the amino acid sequence of SEQ ID NO: 4,LCDR1-PcrV has the amino acid sequence of SEQ ID NO: 6, LCDR2-PcrV hasthe amino acid sequence of SEQ ID NO: 7, and LCDR3-PcrV has the aminoacid sequence of SEQ ID NO: 8. In embodiments, the bispecific antibodyhas a heavy chain and a light chain. The heavy chain includes theformula VH-CH1-H1-L1-S-L2-H2-CH2-CH3, where VH is an anti-P. aeruginosaPcrV heavy chain variable domain including the amino acid sequence ofSEQ ID NO: 1, CH1 is a heavy chain constant region domain-1, H1 is afirst heavy chain hinge region fragment, L1 is a first linker, S is ananti-P. aeruginosa Psl ScFv molecule including the amino acid sequenceof SEQ ID NO: 9, L2 is a second linker, H2 is a second heavy chain hingeregion fragment, CH2 is a heavy chain constant region domain-2, and CH3is a heavy chain constant region domain-3. The light chain includes theformula VL-CL, where VL is an anti-P. aeruginosa PcrV light chainvariable domain including the amino acid sequence of SEQ ID NO: 5 and CLincludes an antibody light chain kappa constant region or an antibodylight chain lambda region. In certain aspects, CH1 can include the aminoacid sequence of SEQ ID NO: 16. In certain aspects L1 and L2 can be thesame or different, and can independently include (a) [GGGGS]n, where nis 0, 1, 2, 3, 4, or 5 (SEQ ID NO: 23), (b) [GGGG]n, where n is 0, 1, 2,3, 4, or 5 (SEQ ID NO: 24), or a combination of (a) and (b). In certainaspects, H1 can include the amino acid sequence EPKSC (SEQ ID NO: 17).In certain aspects, H2 can include the amino acid sequence DKTHTCPPCP(SEQ ID NO: 18). In certain aspects, CH2-CH3 can include the amino acidsequence of SEQ ID NO: 19. In certain aspects CH2-CH3 can include theamino acid sequence of SEQ ID NO: 20. In certain aspects, the heavychain of the bispecific antibody can include the amino acid sequence ofSEQ ID NO: 21, and the light chain includes the amino acid sequence ofSEQ ID NO: 22.

Pharmaceutical Compositions Comprising Anti-Pseudomonas aeruginosa Psland PcrV Bispecific Binding Molecules

Pharmaceutical compositions used in this disclosure comprisepharmaceutically acceptable carriers well known to those of ordinaryskill in the art. Preparations for parenteral administration includesterile aqueous or non-aqueous solutions, suspensions, and emulsions.

The amount of an anti-Pseudomonas aeruginosa Psl and PcrV bispecificantibody or fragment, variant or derivative thereof that can be combinedwith the carrier materials to produce a single dosage form will varydepending upon the subject treated and the particular mode ofadministration. Exemplary dosage regimens include a single intravenousinfusion of an anti-Pseudomonas aeruginosa Psl and PcrV bispecificantibody of about 500 to about 3000 mg, e.g., about 500 mg, 600, 700,750, 1000, 1500 or about 3000 mg. Dosage regimens also can be adjustedto provide the optimum desired response (e.g., a prophylactic responseor a therapeutic treatment response).

The route of administration of an anti-Pseudomonas aeruginosa Psl andPcrV bispecific antibody or fragment, variant or derivative thereof, canbe, for example, parenteral. The term parenteral as used hereinincludes, e.g., intravenous, intraarterial, intraperitoneal,intramuscular, or subcutaneous administration. A suitable form foradministration would be a solution for injection, in particular forintravenous or intraarterial injection or drip.

Anti-Pseudomonas aeruginosa Psl and PcrV bispecific antibodies orfragments, variants or derivatives thereof can be administered in apharmaceutically effective amount for the in vivo treatment of orprevention of Pseudomonas aeruginosa infection. In this regard, it willbe appreciated that the disclosed binding molecules can be formulated soas to facilitate administration and promote stability of the activeagent.

In keeping with the scope of the disclosure, anti-Pseudomonas aeruginosaPsl and PcrV bispecific antibodies or fragments, variants or derivativesthereof, can be administered to a human or other animal in accordancewith the aforementioned methods of treatment in an amount sufficient toproduce a therapeutic effect. The anti-Pseudomonas aeruginosa Psl andPcrV bispecific antibodies or fragments, variants or derivatives thereofdisclosed herein can be administered to such human or other animal in aconventional dosage form prepared by combining the antibody of thedisclosure with a conventional pharmaceutically acceptable carrier ordiluent according to known techniques.

Effective doses of the compositions of the present disclosure fortreatment of Pseudomonas aeruginosa infection vary depending upon manydifferent factors, including means of administration, target site,physiological state of the patient, whether the patient is human or ananimal, other medications administered, and whether treatment isprophylactic or therapeutic. Usually, the patient is a human butnon-human mammals including transgenic mammals can also be treated.Treatment dosages can be titrated using routine methods known to thoseof skill in the art to optimize safety and efficacy.

Anti-Pseudomonas aeruginosa Psl and PcrV bispecific antibodies orfragments, variants or derivatives thereof can be administered multipleoccasions at various frequencies, e.g., as a single dose, depending onvarious factors known to those of skill in the art.

Methods for Preventing or Treating Nosocomial Infections

This disclosure provides a method of preventing nosocomial infection ina susceptible human subject, where the method includes administering tothe subject a dose of about 500 mg to about 3000 mg, e.g., 500 mg, 600mg, 700 mg, 750 mg, 1000 mg, 1500 mg or 3000 mg, of a bispecificantibody that specifically binds Pseudomonas aeruginosa Psl and PcrV.The bispecific antibody can be administered, e.g., as a single dose ofabout 500 mg to about 3000 mg, e.g., 500 mg, 600 mg, 700 mg, 750 mg,1000 mg, 1500 mg, or 3000 mg. In certain aspects the bispecific antibodycan be administered as an IV infusion. A susceptible human subject, asdescribed in more detail elsewhere herein, is a person who is at risk ofcontracting a nosocomial infection but at the time of administrationdoes not have an infection or shows no symptoms of an infection; or aperson who has contracted a nosocomial infection that requiresintervention or mitigation. The method further includes monitoring thesubject for symptoms following administration of the bispecific antibodyfor, e.g., through 1 day, 3 days, 5 days, 7 days, 10 days, 15 days, 21days, 28 days, or 30 days. In an embodiment, the method includesmonitoring the subject for symptoms through at least about 21 days ormore from the day of administration. “Nosocomial infections” are definedelsewhere herein and include, e.g., pneumonia, bacteremia, boneinfection, joint infection, skin infection, burn infection, woundinfection, peritonitis, sepsis, and/or an abscess. Symptoms associatedwith nosocomial infections, e.g., pneumonia, are known in the art, andexemplary symptoms are described in the Examples below. In certainaspects the nosocomial infection is caused by, or is exacerbated by P.aeruginosa. According to this method, the human subject is successfullytreated if, e.g., at 1 day, 3 days, 5 days, 8 days, 10 days, 15 days, 21days, 28 days, or 30 days post-administration, the subject remainssymptom-free (if the subject was symptom free at the time ofadministration) or displays less severe symptoms than would be expectedif not treated with MEDI3902. In an embodiment, the human subject issuccessfully treated if at 21 days post-administration, the subjectremains symptom-free (if the subject was symptom free at the time ofadministration) or displays less severe symptoms than would be expectedif not treated with MEDI3902. In another embodiment, the human subjectis successfully treated if at 28 or 30 days post-administration, thesubject remains symptom-free (if the subject was symptom free at thetime of administration) or displays less severe symptoms than would beexpected if not treated with MEDI3902.

In another aspect this disclosure provides a method of preventing ortreating pneumonia, e.g., hospital acquired or not hospital acquiredpneumonia in a susceptible human subject, where the method includesadministering to the subject a dose of about 500 mg to about 3000 mg,e.g., 500 mg, 600 mg, 700 mg, 750 mg, 1000 mg, 1500 mg or 3000 mg, of abispecific antibody that specifically binds Pseudomonas aeruginosa Psland PcrV. The bispecific antibody can be administered e.g., as a singledose of about 500 mg to about 3000 mg, e.g., 500 mg, 600 mg, 700 mg, 750mg, 1000 mg, 1500 mg or 3000 mg. In certain aspects the bispecificantibody can be administered as an IV infusion. In certain aspects thepneumonia is nosocomial or iatrogenic. A susceptible human subject, asdescribed in more detail elsewhere herein, is a person who is at risk ofcontracting pneumonia but at the time of administration does not havepneumonia symptoms; or a person who has contracted pneumonia thatrequires intervention or mitigation. The method further includesmonitoring the subject for pneumonia symptoms following administrationof the bispecific antibody for, e.g., through 1 day, 3 days, 5 days, 7days, 10 days, 15 days, 21 days, 28 days, or 30 days. In an embodiment,the method includes monitoring the subject for symptoms at least about21 days from the day of administration. Symptoms associated withpneumonia are known in the art, and exemplary symptoms are described inthe Examples below. In certain aspects the pneumonia is caused by, or isexacerbated by, P. aeruginosa. According to this method, the humansubject is successfully treated if, e.g., at 1 day, 3 days, 5 days, 8days, 10 days, 15 days, 21 days, 28 days, or 30 dayspost-administration, the subject remains symptom-free (if the subjectwas symptom free at the time of administration) or displays less severesymptoms than would be expected if not treated with MEDI3902. In anembodiment, the human subject is successfully treated if at 7 dayspost-administration, the subject remains symptom-free (if the subjectwas symptom-free at the time of administration) or displays less severesymptoms than would be expected if not treated with MEDI3902. In anembodiment, the human subject is successfully treated if at 21 dayspost-administration, the subject remains symptom-free (if the subjectwas symptom-free at the time of administration) or displays less severesymptoms than would be expected if not treated with MEDI3902. In anotherembodiment, the human subject is successfully treated if at 28 or 30days post-administration, the subject remains symptom-free (if thesubject was symptom free at the time of administration) or displays lesssevere symptoms than would be expected if not treated with MEDI3902.

In another aspect this disclosure provides a method of preventing ortreating a disease caused by Pseudomonas aeruginosa, e.g., pneumonia,tracheobronchitis, bacteremia, endocarditis, meningitis, otitis media,bacterial keratitis, endophthalmitis, osteomyelitis, gastrointestinaldisease, skin infection, septicemia, or any combination thereof, in asusceptible human subject, where the method includes administering tothe subject a dose of about 500 mg to about 3000 mg, e.g., 500 mg, 600mg, 700 mg, 750 mg, 1000 mg, 1500 mg, or 3000 mg, of a bispecificantibody that specifically binds Pseudomonas aeruginosa Psl and PcrV.The bispecific antibody can be administered, e.g., as a single dose ofabout 500 mg to about 3000 mg, e.g., 500 mg, 600 mg, 700 mg, 750 mg,1000 mg, 1500 mg, or 3000 mg. In certain aspects the bispecific antibodycan be administered as an IV infusion. In certain aspects the diseasecaused by Pseudomonas aeruginosa is nosocomial or iatrogenic. Asusceptible human subject, as described in more detail elsewhere herein,is a person who is at risk of contracting a disease treatable orpreventable by the methods provided herein but at the time ofadministration does not have disease symptoms; or a person who hascontracted disease caused by Pseudomonas aeruginosa that requirestreatment, intervention or mitigation. The method further includesmonitoring the subject for disease symptoms following administration ofthe bispecific antibody through, e.g., 1 day, 3 days, 5 days, 8 days, 10days, 15 days, 21 days, 28 days, or 30 days. In an embodiment, themethod includes monitoring the subject for symptoms through at leastabout 21 days from the day of administration. Symptoms associated withpneumonia are known in the art, and exemplary symptoms are described inthe Examples below. According to this method, the human subject issuccessfully treated if, e.g., at 1 day, 3 days, 5 days, 8 days, 10days, 15 days, 21 days, 28 days, or 30 days post-administration, thesubject remains symptom-free (if the subject was symptom free at thetime of administration) or displays less severe symptoms than would beexpected if not treated with MEDI3902. In an embodiment, the humansubject is successfully treated if at 7 days post-administration, thesubject remains symptom-free (if the subject was symptom-free at thetime of administration) or displays less severe symptoms than would beexpected if not treated with MEDI3902. In an embodiment, the humansubject is successfully treated if at 21 days post-administration, thesubject remains symptom-free (if the subject was symptom free at thetime of administration) or displays less severe symptoms than would beexpected if not treated with MEDI3902. In another embodiment, the humansubject is successfully treated if at 28 or 30 days post-administration,the subject remains symptom-free (if the subject was symptom free at thetime of administration) or displays less severe symptoms than would beexpected if not treated with MEDI3902.

In certain aspects the bispecific antibody to be administered accordingto the methods herein includes a binding domain which specifically bindsto Psl comprising a set of Complementarity-Determining Regions (CDRs):HCDR1-Psl, HCDR2-Psl, HCDR3-Psl, LCDR1-Psl, LCDR2-Psl, and LCDR3-Psl,wherein HCDR1-Psl has the amino acid sequence of SEQ ID NO: 10,HCDR2-Psl has the amino acid sequence of SEQ ID NO: 11, HCDR3-Psl hasthe amino acid sequence of SEQ ID NO: 12, LCDR1-Psl has the amino acidsequence of SEQ ID NO: 13, LCDR2-Psl has the amino acid sequence of SEQID NO: 14, and LCDR3-Psl has the amino acid sequence of SEQ ID NO: 15;and a binding domain which specifically binds to PcrV comprisingHCDR1-PcrV, HCDR2-PcrV, HCDR3-PcrV, LCDR1-PcrV, LCDR2-PcrV, andLCDR3-PcrV, wherein HCDR1-PcrV has the amino acid sequence of SEQ ID NO:2, HCDR2-PcrV has the amino acid sequence of SEQ ID NO: 3, HCDR3-PcrVhas the amino acid sequence of SEQ ID NO: 4, LCDR1-PcrV has the aminoacid sequence of SEQ ID NO: 6, LCDR2-PcrV has the amino acid sequence ofSEQ ID NO: 7, and LCDR3-PcrV has the amino acid sequence of SEQ ID NO:8. In embodiments, the bispecific antibody has a heavy chain and a lightchain. The heavy chain includes the formulaVH-CH1-H1-L1-S-L2-H2-CH2-CH3, where VH is an anti-PcrV heavy chainvariable domain including the amino acid sequence of SEQ ID NO: 1, CH1is a heavy chain constant region domain-1, H1 is a first heavy chainhinge region fragment, L1 is a first linker, S is an anti-Psl ScFvmolecule including the amino acid sequence of SEQ ID NO: 9, L2 is asecond linker, H2 is a second heavy chain hinge region fragment, CH2 isa heavy chain constant region domain-2, and CH3 is a heavy chainconstant region domain-3. The light chain includes the formula VL-CL,where VL is an anti-PcrV light chain variable domain including the aminoacid sequence of SEQ ID NO: 5 and CL includes an antibody light chainkappa constant region or an antibody light chain lambda region. Incertain aspects, CH1 can include the amino acid sequence of SEQ ID NO:16. In certain aspects L1 and L2 can be the same or different, and canindependently include (a) [GGGGS]n, where n is 0, 1, 2, 3, 4, or 5 (SEQID NO: 23), (b) [GGGG]n, where n is 0, 1, 2, 3, 4, or 5 (SEQ ID NO: 24),or a combination of (a) and (b). In certain aspects, H1 can include theamino acid sequence EPKSC (SEQ ID NO: 17). In certain aspects, H2 caninclude the amino acid sequence DKTHTCPPCP (SEQ ID NO: 18). In certainaspects, CH2-CH3 can include the amino acid sequence of SEQ ID NO: 19.In certain aspects CH2-CH3 can include the amino acid sequence of SEQ IDNO: 20. In certain aspects, the heavy chain of the bispecific antibodycan include the amino acid sequence of SEQ ID NO: 21, and the lightchain includes the amino acid sequence of SEQ ID NO: 22.

The bispecific antibody for use in the methods provided herein can beadministered, e.g., as a single dose of about 500 mg to about 3000 mg,e.g., 500 mg, 600 mg, 700 mg, 750 mg, 1000 mg, 1500 mg, or 3000 mg.

In some embodiments, the serum target level of the bispecific antibodyor antigen-binding fragments thereof is in the range of about 1 μg/mL toabout 10 μg/mL, about 2 μg/mL to about 9 μg/mL, about 3 μg/mL to about 8μg/mL, about 4 μg/mL to about 7 μg/mL, about 5 μg/mL to about 6 μg/mL,about 5 μg/mL to about 7 μg/mL, 5 μg/mL to about 8 μg/mL, 5 μg/mL toabout 9 μg/mL, or about 5 μg/mL to about 10 μg/mL. In other embodiments,the serum target level of the bispecific antibody or antigen-bindingfragments thereof is in the range of about 1 μg/mL to about 6 μg/mL,about 1 μg/mL to about 5 μg/mL, about 1 μg/mL to about 4 μg/mL, about 1μg/mL to about 3 μg/mL, or about 1 μg/mL to about 2 μg/mL. In oneembodiment, the serum target level of the bispecific antibody orantigen-binding fragments thereof is at least 1.7 μg/mL. In anotherembodiment, the serum target level of the bispecific antibody orantigen-binding fragments thereof is at least 5.3 μg/mL. A serum targetlevel of about 1.7 μg/mL is expected to prevent or treat infectionscaused by >80-90% of P. aeruginosa strains. A serum target level ofabout 5.3 μg/mL is expected to prevent or treat infections caused by thetoughest P. aeruginosa strains (e.g., those strains that are highlyvirulent and resistant to antibiotics, such as ARC3928 and ARC3502.)

In certain aspects the bispecific antibody for use in the methodsprovided herein can be administered as an IV infusion.

The methods provided herein are suitable for use with susceptible humansubjects as described elsewhere herein. Examples include subjects whoare about to be hospitalized, are currently hospitalized, were recentlyhospitalized, are about to be, currently, or recently on a mechanicalventilator, or a combination thereof. Hospitalization, in someinstances, can be in an intensive care unit (ICU). Mechanicalventilation, if required, can be through intubation, e.g., through anendotracheal or nasotracheal tube, or through a tracheostomy. Patientswho are about to be, are currently, or were recently on mechanicalventilation can have a heightened risk of contracting a respiratoryinfection, e.g., pneumonia, e.g., Pseudomonas aeruginosa pneumonia. Inthose situations where mechanical ventilation is indicated,administration of a bispecific antibody as provided by the disclosedmethods can reduce the risk of contracting pneumonia, for example, whilecurrently on mechanical ventilation, after mechanical ventilation is nolonger required, or a combination thereof.

In certain aspects the subject is colonized with Pseudomonas aeruginosain the respiratory tract, e.g., the lower respiratory tract, at the timeof administration of the bispecific antibody. In certain aspects thesubject's respiratory tract is colonized with P. aeruginosa one, two,three, or four days prior to administration of the bispecific antibody.Colonization can be measured, e.g., by detection of P. aeruginosa in atracheal aspirate within 6 hours, 12 hours, 24 hours, 48 hours, 72hours, or 96 hours prior to administration of the bispecific antibody.In certain aspects of the methods provided herein, the subject has notreceived antibiotics considered active against the P. aeruginosa strainwith which the subject is colonized prior to administration of thebispecific antibody. In certain aspects, the subject's respiratory tractcan be additionally colonized by Staphylococcus aureus at the time ofadministration of the bispecific antibody.

In certain aspects the subject does not have pneumonia symptoms at thetime of administration of the bispecific antibody. Symptoms can bemeasured according to the Clinical Pulmonary Infection Score (CPIS). Alack of symptoms can be inferred e.g., if at 24 hours prior to theadministration of the bispecific antibody the subject has a CPIS of lessthan 6.

The methods provided herein include monitoring a subject for diseasesymptoms, e.g., pneumonia symptoms, following administration of thebispecific antibody. In certain aspects, the subject can be monitoredfor pneumonia by chest x-ray, observation of respiratory signs orsymptoms of pneumonia, microbiologic confirmation of pneumonia, or anycombination thereof. A subject can be determined to have pneumonia,e.g., when a new or worsening infiltrate consistent with pneumonia isobserved on a chest x-ray, when the subject displays at least two minoror at least one major respiratory sign or symptoms of pneumonia, when aspecimen obtained from the subject is positive for P. aeruginosa byculture, or any combination thereof. In certain aspects the specimen isa respiratory secretion of the subject. A respiratory secretion can beobtained from expectorated sputum, by endotracheal aspiration, bybronchoscopy with bronchoalveolar lavage, by use of a protected-specimenbrush sampling in an intubated subject, or any combination thereof.

Minor respiratory signs or symptoms of pneumonia include, withoutlimitation, a body temperature of greater than about 38° C., a core bodytemperature of less than about 35° C., a white blood cell count ofgreater than about 10,000 cells per cubic millimeter (mm³), a whiteblood cell count of less than about 4,500 cells per mm³, a bandneutrophil count of greater than about 15%, production of new purulentendotracheal secretions or sputum, new auscultatory findings, dullnessto percussion, a new onset of cough, dyspnea, tachypnea, hypoxemia, orany combination thereof. Major respiratory signs or symptoms ofpneumonia can include, without limitation, an acute change made in theventilatory support system to enhance oxygenation comprising a PaO₂/FiO₂ratio less than about 240 mm Hg maintained for at least four hours, adecrease in the PaO₂/FiO₂ ratio of greater than about 50 mm Hgmaintained for at least four hours, the necessity to initiate orreinitiate mechanical ventilation in a non-mechanically ventilatedsubject, or any combination thereof. Microbiologic confirmation ofpneumonia can include, without limitation, a respiratory specimenpositive for P. aeruginosa by culture, a blood culture positive for P.aeruginosa, a pleural fluid aspirate or lung tissue culture positive forP. aeruginosa, or any combination thereof.

In certain aspects, the methods provided by this disclosure can furtherinclude administering an antibiotic to the subject prior to,concurrently with, and/or following administration of the bispecificantibody. Suitable antibiotics can include, without limitation,aminoglycosides, ticarcillin, ureidopenicillins, ciprofloxacin,cefepime, gentamicin, amikacin, tobramycin, ceftazidime, aztreonam,cefotaxime, or any combination thereof. Suitable dosages and length oftreatment can be readily determined by a healthcare provider. In certainaspects, the P. aeruginosa strain with which the subject is colonized issensitive to the antibiotic chosen for administration. In other aspects,however, the P. aeruginosa strain with which the subject is colonized isresistant or partially resistant to one or more of the availableantibiotics chosen for administration. In preclinical studies, forexample, MEDI3902 has shown synergistic enhancement of antibiotictherapy against P. aeruginosa pneumonia with distinct antibiotic classesagainst both antibiotic-sensitive and antibiotic-resistant P.aeruginosa.

In certain aspects, the methods provided by this disclosure can furtherinclude administering an antihistamine to the subject prior to,concurrently with, and/or following administration of the bispecificantibody. Suitable antihistamines can include, without limitation,diphenhydramine, clemastine, dexchlorpheniramine, azelastine,cetirizine, desloratadine, fexofenadine, levocetirizine, loratadine,olopatadine, or any combination thereof. Suitable dosages and length oftreatment can be readily determined by a healthcare provider.

In certain aspects, the methods provided by this disclosure includemonitoring a subject for serum pharmacokinetics of the bispecificantibody, tracheal secretion pharmacokinetics of the bispecificantibody, or a combination thereof. A subject can maintain, e.g., aserum level of the bispecific antibody of at least about 1 μg/mL, atleast about 2 μg/mL, at least about 3 μg/mL, at least about 4 μg/mL, atleast about 5 μg/mL, or at least about 5.3 μg/mL or more through day 7,day 14, day 21, day 28, day 30, day 35, day 42, or day 49 followingadministration of the bispecific antibody. For example, a subject canmaintain a serum level of the bispecific antibody of at least about 1.7μg/mL, or at least about 5.3 μg/mL, or more through day 7, day 14, day21, day 28, day 30, day 35, day 42, or day 49 following administrationof the bispecific antibody. Based on observed increased clearance in ICUpatients for monoclonal antibodies directed at bacterial pathogens(e.g., MEDI4893), pharmacokinetics were modelled for ICU patients withmechanical ventilation following a single dose of MEDI3902. A singledose of 1500 mg MEDI3902 is predicted to maintain serum exposure atabout 1 μg/mL for ≥21 days in >90% subjects. A single dose of 3000 mgMEDI3902 is predicted to maintain serum exposure at about 1.7 μg/mL for≥21 days in >90% subjects. Based on pharmacokinetics in healthyvolunteers (e.g., without increased clearance like that observed in ICUpatients), a single dose of 500 mg, 1500 mg or 3000 mg is expected tomaintain serum exposure at about 1 μg/mL for ≥21 days in >90% subjects,for example 1.7 μg/mL for ≥21 days in >90% subjects, such as about 5.3μg/mL for >21 days in >90% subjects. Of course, the serum level of thebispecific antibody will vary throughout the monitoring period, and canreach a maximal concentration of the bispecific antibody, or C_(max), ofabout 100 μg/mL, about 200 μg/mL, about 300 μg/mL, about 400 μg/mL,about 500 μg/mL, or more than about 600 μg/mL, depending on the dose ofbispecific antibody administered. In certain aspects, the area under theconcentration-time curve from time zero to infinity (AUC_(∞)) can bemeasured. For example, the AUC_(∞) can be, e.g., about 2000 μg·day/mL toabout 6000 μg·day/mL, e.g., about 4000 μg·day/mL for a 1500 mg dose ofthe bispecific antibody. In certain aspects, serum terminal half-lifecan be measured. In certain aspects the volume of distribution at steadystate (Vd_(ss)) can be measured. The subject can be monitored forpharmacokinetic parameters through day 7, day 14, day 21, day 28, day30, day 35, day 42, day 49, or longer following administration of thebispecific antibody. In certain aspects the subject can be monitoredthrough day 21. In certain aspects the subject can be monitored throughday 49.

The following disclosed embodiments are merely representative. Thus,specific structural, functional, and procedural details disclosed in thefollowing examples are not to be interpreted as limiting.

EXAMPLES Example 1: Clinical Trial Phase 1 Study

A Phase 1, first time in human, randomised, double-blind,placebo-controlled, dose-escalation study was conducted that evaluatedthe safety and tolerability of a single ascending IV dose of MEDI3902 inhealthy adult subjects. Adverse event (AE), PK, and ADA data werecollected through the last study visit (60 days postdose/Day 61) in the42 subjects who received MEDI3902 at doses of 250 mg (n=3), 750 mg(n=15), 1500 mg (n=15), or 3000 mg (n=9) and 14 subjects who receivedplacebo. All of the AEs in both the MEDI3902 and placebo groups wereeither Grade 1 or Grade 2 in severity. The most frequently reported AEswere Grade 1 or Grade 2 infusion-related reactions (15/42 subjects[35.7%] in the total MEDI3902 group; none in the placebo group) andGrade 1 or Grade 2 headache (6/42 subjects [14.3%] in the total MEDI3902group; 2/14 subjects [14.3%] in the placebo group). Adverse events ofspecial interest (AESIs) included the events of infusion-relatedreaction (15/42 subjects [35.7%] in the total MEDI3902 group; none inthe placebo group), which were mitigated by treatment withdiphenhydramine and by slowing the infusion rate. Most events ofinfusion-related reaction resolved either during or immediately afterthe infusion or by the next day. Two subjects (1 subject in the 750 mgMEDI3902 dose group and 1 subject in the 3000 mg dose group) did notcomplete dosing due to infusion-related reactions. No serious adverseevents (SAEs) were reported.

PK and immunogenicity of MEDI3902 were studied in healthy adultvolunteers in the Phase 1 study. Blood samples were collected predoseand at various time points up to 60 days postdose for quantitatingMEDI3902 concentrations in serum and for detecting ADA against MEDI3902.Following IV infusion, MEDI3902 exhibited linear PK in healthy adultsubjects. Serum peak concentrations were observed immediatelypostinfusion, and declined in a bi-exponential manner over time.MEDI3902 concentrations in the 250 mg dose cohort were measurable up to42 days postdose while other high-dose cohorts had measurable serumconcentration through 60 days postdose. A single dose of 750 mg IVmaintained serum exposure above the target level of 5.3 μg/mL for 28days for most subjects in this cohort. MEDI3902 exposure in serumincreased in an approximately dose-proportional manner from 250 mg to1500 mg, and in a slightly less than dose proportional manner from 1500mg to 3000 mg. C_(max) increased from 100 μg/mL at 250 mg to 468 μg/mLat 1500 mg and to 838 μg/mL at 3000 mg. The area under theconcentration-time curve from time zero to infinity (AUC_(∞)) increaseddose-proportionally from 694 μg·day/mL at 250 mg to 4246 μg·day/mL at1500 mg. AUC_(∞) at 3000 mg was 6540 μg·day/mL. Serum terminal half-lifewas estimated to be 7-9 days. Volume of distribution at steady state(Vd_(ss)) was 3.4-4.9 L, indicating limited distribution into theextravascular space. Of the 42 subjects, 1 subject in Cohort 4 (3000 mg)tested ADA positive postdose on Day 61. The serum concentration ofMEDI3902 in this subject rapidly decreased 45 days postdose and droppedto a level below the lower limit of quantification (LLOQ) 60 dayspostdose, suggesting an impact of ADA on MEDI3902 PK.

MEDI3902 was found to be generally safe in the initial Phase 1 study.

Example 2: Clinical Trial Phase 2b Study

In the Phase 2, randomized, double-blind, placebo-controlled,single-dose, dose-ranging, proof-of-concept, approximately 429 subjectswill be enrolled and dosed at approximately 120 sites primarily inEurope. Subjects will be randomly assigned in a 1:1:1 ratio to receive asingle IV dose of 1500 mg or 3000 mg MEDI3902, or placebo. Dosing schemecan also be changed to a 1:1 randomization between single IV dose ofMEDI3902 at 1500 mg (or 3000 mg) or placebo. Randomization will bestratified by geographic region and by whether subjects received anti-P.aeruginosa antibiotic treatment (duration of ≤72 hours) within the 96hours prior to randomization. Subjects will be followed through Day 50(49 days post investigational product administration) (FIG. 1).

In order to be enrolled, subjects are required to be 18 years of age orolder, in the Intensive Care Unit, currently intubated and mechanicallyventilated, and expected to remain intubated and mechanically ventilatedfor at least three days at the time of study entry. Subject must also becolonized with P. aeruginosa in the lower respiratory tract, asdemonstrated by having a tracheal sample positive for P. aeruginosawithin 36 hours prior to randomization, but are currently free of P.aeruginosa-related disease. Subjects with evidence of resolved pneumoniacan be enrolled.

Subjects are not eligible to participate if they have acute confirmed orsuspected Pseudomonas disease at enrollment or before receivingMEDI3902. Subjects are also not eligible to participate if they havepreviously received a monoclonal antibody, if they have a clinicalpulmonary infection score (CPIS) of six or greater within the past 24hours prior to MEDI3902 dosing, or if they have an Acute Physiology andChronic Health Evaluation-II (APACHE-II) score of greater than or equalto 25 or a SOFA score of greater than or equal to 12 at time ofrandomization. Subjects with active pulmonary disease that would impairthe ability to diagnose pneumonia (e.g., active tuberculosis or fungaldisease, obstructing lung cancer, large pleural effusion or empyema,cystic fibrosis, or acute respiratory distress syndrome with lung “whiteout”) are not eligible. Subjects who are tracheostomy-dependent prior tohospital admission and subjects with burns on more than 40% of bodysurface are not eligible. In addition, subjects are not eligible if theyreceived an anti-P. aeruginosa antibiotic for more than 72 hours within96 hours prior to randomization, wherein the antibiotic is consideredactive against the P. aeruginosa strain with which the subject iscolonized, or wherein ongoing receipt of the anti-P. aeruginosaantibiotic is anticipated.

Subjects will be randomly assigned to receive a single dose of 1500 mgMEDI3902, 3000 mg MEDI3902, or placebo administered via IV infusion onDay 1. All subjects will be premedicated with antihistamine, forexample, 50 mg diphenhydramine IV, clemastine 2 mg IV, ordexchlorpheniramine 5 mg IV (or another antihistamine preparationutilized in routine clinical practice for management of acute allergicreactions) within 15-30 minutes prior to start of study medicationinfusion. Additional premedication of subjects with acetaminophen 650 mgorally and/or methylprednisolone 20 mg IV in combination with theantihistamine, or the antihistamine together with famotidine 20 mgorally with or without acetaminophen and/or methylprednisolone prior tostart of investigational product infusion, can be authorized.

The primary efficacy endpoint for this study is the incidence ofnosocomial pneumonia caused by P. aeruginosa through 21 days postdose(Day 22) after a single dose of MEDI3902 in mechanically ventilatedsubjects at risk for P. aeruginosa nosocomial pneumonia. The dosagelevels were selected based on data from the Phase 1 study (see Example1), PK/PD data from preclinical pharmacology studies (e.g., EC₉₀ in amurine P. aeruginosa-induced lethal pneumonia model), PK modelling basedon observed increased clearance in ICU patients of other monoclonalantibodies directed at bacterial pathogens (FIG. 2), and a clinicalreport of median (range) time to VAP onset of 10 days. Some mechanicallyventilated subjects will continue to require mechanical ventilationthroughout the 21-day postdose period, but some subjects may be weanedoff the ventilator during this period. Since these latter subjects maydevelop nosocomial pneumonia caused by P. aeruginosa after they nolonger require mechanical ventilation, primary efficacy will beevaluated in subjects who were on mechanical ventilation at the time ofenrolment, regardless of whether they remain on or are weaned offmechanical ventilation during the 21-day postdose period. The secondaryefficacy endpoints will evaluate separately the incidence of nosocomialpneumonia caused by P. aeruginosa while on mechanical ventilation andthe incidence of nosocomial pneumonia caused by P. aeruginosa aftermechanical ventilation is no longer required through 21 days postdose.

The primary safety endpoints will assess TEAEs, TESAEs, and AESIsthrough 49 days postdose.

The secondary endpoints (MEDI3902 serum concentration, PK parameters,and ADA response to MEDI3902 through 49 days postdose) are designed toassess the presence of MEDI3902 in vivo.

Data analysis will be performed after all subjects have completed thestudy. Analysis of efficacy, pharmacokinetic (PK), anti-drug antibody(ADA), and safety will be performed on all data collected after the lastsubject has completed follow-up through 49 days postdose to demonstratethat a single IV dose of 1500 mg (and possibly 3000 mg) of MEDI3902 hasan acceptable safety profile and is capable of reducing the incidence ofP. aeruginosa pneumonia in mechanically ventilated subjects in theIntensive Care Unit (ICU) who are colonized with P. aeruginosa in thelower respiratory tract through 21 days postdose (irrespective ofmechanical ventilation at the time of diagnosis).

Tracheal aspirate samples are collected for microbiological assessmentof P. aeruginosa colonization on Days 2, 4 (±1 day), 8 (±1 day), 15 (±1day), 29 (±1 day), and 50 (±1 day), provided the subject remainsintubated.

Subjects are monitored for clinical symptoms of pneumonia and otherserious P. aeruginosa infection daily while in hospital, and inaccordance with symptoms after hospital discharge. At these times, themonitoring includes a physical examination and evaluation of vitalsigns. While in the hospital, CPIS is assessed daily while the subjectremains on mechanical ventilation.

For subjects with suspected serious P. aeruginosa infection, clinicalsymptoms (CPIS, SOFA, physical exam, and vital signs) are assessed atthe day of onset and then daily through resolution. Blood samples areanalyzed in all subjects with suspected serious P. aeruginosa infectionon the day of onset and the two following days. Blood sampling analysesare repeated every other day in those that are positive for P.aeruginosa pneumonia until they are negative for P. aeruginosa. Trachealor bronchial aspirates are analyzed in subjects with suspected seriousP. aeruginosa infection that are intubated on the day of onset and thetwo following days. Tracheal or bronchial aspirates analyses arerepeated every day in those that are positive for P. aeruginosapneumonia until resolution. Expectorated sputum is analyzed in subjectswith suspected serious P. aeruginosa infection that are not intubated(unless bronchoscopy performed for clinical management and BAL or PSBsample available) on the day of onset and the two following days.Expectorated sputum analyses are repeated every other day in those thatare positive for P. aeruginosa pneumonia until resolution. Chest X-raysare performed on subjects with suspected serious P. aeruginosa infectionon the day of onset. Chest X-rays are repeated in those with confirmedpneumonia as clinically indicated through resolution.

In subjects who are mechanically ventilated at the time of diagnosis ofP. aeruginosa pneumonia, the criteria for the diagnosis requires thatthe subject demonstrate the following radiographic, clinical, andmicrobiologic new onset symptoms/signs that are not due to any othernon-infectious cases.

1. Radiographic criteria:

-   -   a. New or worsening infiltrate consistent with pneumonia on        chest x-ray obtained within 24 hours of the event (diagnosed by        a qualified radiologist)

AND

2. Clinical criteria: At least 2 of the following minor or 1 majorrespiratory sign or symptom of new onset:Minor criteria:

-   -   b. Systemic signs of infection (one or more of the following):        Abnormal temperature (oral or tympanic temperature>38° C. or a        core temperature≥38.3° C. or hypothermia, defined as a core body        temperature of <35° C.), and/or abnormal WBC count (WBC        count>10,000 cells/mm³, WBC count<4,500 cells/mm³, or >15% band        neutrophils)    -   c. Production of new purulent endotracheal secretions    -   d. New physical examination findings consistent with        pneumonia/pulmonary consolidation such as auscultatory findings        (eg, rales, rhonchi, bronchial breath sounds) or dullness to        percussion        Major criteria:    -   e. Acute changes made in the ventilatory support system to        enhance oxygenation, as determined by:        -   i. PaO₂/FiO₂ ratio<240 mm Hg maintained for at least 4 hours            OR        -   ii. A decrease in PaO₂/FiO₂ by ≥50 mm Hg maintained for at            least 4 hours

AND

3. Microbiologic confirmation (obtained within 24 hours of onset of theevent): At least 1 of the following:

-   -   f. Respiratory specimen positive for P. aeruginosa by culture        includes a specimen of respiratory secretion obtained by        endotracheal aspiration or by bronchoscopy with BAL or PSB        sampling in intubated subjects. In subjects who are not        intubated but meet the protocol definition of mechanical        ventilation, a specimen of expectorated sputum would be        acceptable.    -   g. Blood culture positive for P. aeruginosa (and no apparent        primary source of infection outside the lung)    -   h. Pleural fluid aspirate or lung tissue culture positive for P.        aeruginosa during episode of pneumonia (only if obtained as part        of the subject's necessary clinical management).

Subjects are considered mechanically ventilated if they are intubatedwith an endotracheal or nasotracheal tube and are receiving positivepressure ventilation support or if they are not intubated with anendotracheal or nasotracheal tube, but require 8 or more hours ofpositive pressure ventilation within the past 24 hours.

In subjects who are not mechanically ventilated at the time of diagnosisof P. aeruginosa pneumonia, the criteria for the diagnosis requires thatthe subject demonstrate the following radiographic, clinical, andmicrobiologic new onset symptoms/signs that are not due to any othernon-infectious cases.

4. Radiographic criteria:

-   -   i. New or worsening infiltrate consistent with pneumonia on        chest x-ray obtained within 24 hours of the event (diagnosed by        qualified radiologist)

AND

5. Clinical criteria: At least 2 of the following minor or 1 majorrespiratory sign or symptom of new onset:Minor criteria:

-   -   j. Systemic signs of infection (one or more of the following):        Abnormal temperature (oral or tympanic temperature>38° C. or a        core temperature≥38.3° C. or hypothermia, defined as a core body        temperature of <35° C.), and/or abnormal WBC count (WBC        count>10,000 cells/mm³, WBC count<4,500 cells/mm³, or >15% band        neutrophils)    -   k. A new onset of cough (or worsening of cough)        1. Production of purulent sputum    -   m. New physical examination findings consistent with        pneumonia/pulmonary consolidation such as auscultatory findings        (eg, rales, rhonchi, bronchial breath sounds), dullness to        percussion, or pleuritic chest pain    -   n. Dyspnea, tachypnea (respiratory rate>30 breaths/minute), or        hypoxemia defined as:        -   iii. Oxygen (O₂) saturation<90% or PaO₂<60 mm Hg on room air            if lower than baseline, OR        -   iv. A need to initiate or increase sustained (≥3 hours)            supplemental 02 to maintain pre-event baseline 02            saturations            Major criteria:    -   o. A need to initiate non-invasive mechanical ventilation or        re-initiate invasive mechanical ventilation because of        respiratory failure or worsening of respiratory status

AND

6. Microbiologic confirmation (obtained within 72 hours of onset of theevent): At least 1 of the following:

-   -   p. Respiratory specimen positive for P. aeruginosa by culture.        Includes either expectorated sputum or (only if obtained as part        of the subject's necessary clinical management) a specimen of        respiratory secretions obtained by bronchoscopy with BAL or PSB        sampling    -   q. Blood culture positive for P. aeruginosa (and no other        apparent primary source of infection outside the lung)    -   r. Pleural fluid aspirate or lung tissue culture positive for P.        aeruginosa (only if obtained as part of the subject's necessary        clinical management).

A subject is not considered to be mechanically ventilated when anendotracheal or nasotracheal tube is not in place and the subject doesnot require positive ventilation support for at least 8 hours.

The incidence of P. aeruginosa pneumonia through 21 days post dose iscalculated to demonstrate that administration of 1500 mg or 3000 mgMEDI3902 reduces the incidence of P. aeruginosa pneumonia. The incidenceof P. aeruginosa pneumonia through 21 days post dose will be compared insubjects on mechanical ventilation and subjects in whom mechanicalventilation is no longer required.

Adverse events and new onset chronic disease are reviewed to demonstratethat an administration of 1500 or 3000 mg MEDI3902 is safe.

Example 3: MEDI3902 Reduces Lethality in Acute Pneumonia Model at LowerBacterial Burden

The in vivo effect of MEDI3902 administration was studied in mice usingan acute pneumonia model. Groups of mice (n=6) were injectedintraperitoneally (IP) with either decreasing concentrations of MEDI3902(1.0 mg/kg, 0.5 mg/kg, or 0.2 mg/kg) or a isotype control IgG antibody(negative control, 0.75 mg/kg). Twenty-four hours after treatment, allmice were infected intranasally with 2×10⁵ CFU Pseudomonas aeruginosastrain 6206 (1×LD₁₀₀). As shown in FIG. 3, all control treated animalssuccumbed to infection by 120 hours post infection. However, MEDI3902showed complete protection across all doses, even at 1×LD₁₀₀, suggestingthat a lower target serum level of 1.7 μg/mL in humans will provideprotection across most strains of P. aeruginosa.

The breadth and scope of the present disclosure should not be limited byany of the above-described exemplary embodiments, but should be definedonly in accordance with the following claims and their equivalents.

1. A method of preventing or treating a Pseudomonas aeruginosa infectionin a susceptible human subject comprising administering to the subjectabout 500 to about 3000 mg of a bispecific antibody that specificallybinds Pseudomonas aeruginosa Psl and PcrV and monitoring the subject forsymptoms for 21 days from the day of administration; wherein at 21 dayspost-administration the subject remains symptom-free or displays lesssevere symptoms relative to a cohort of susceptible human subjectsadministered a placebo; wherein the bispecific antibody comprises abinding domain which specifically binds to P. aeruginosa Psl comprisinga set of complementarity determining regions (CDRs): HCDR1-Psl,HCDR2-Psl, HCDR3-Psl, LCDR1-Psl, LCDR2-Psl, and LCDR3-Psl, whereinHCDR1-Psl has the amino acid sequence of SEQ ID NO: 10, HCDR2-Psl hasthe amino acid sequence of SEQ ID NO: 11, HCDR3-Psl has the amino acidsequence of SEQ ID NO: 12, LCDR1-Psl has the amino acid sequence of SEQID NO: 13, LCDR2-Psl has the amino acid sequence of SEQ ID NO: 14, andLCDR3-Psl has the amino acid sequence of SEQ ID NO: 15; and a bindingdomain which specifically binds to P. aeruginosa PcrV comprising a setof CDRs: HCDR1-PcrV, HCDR2-PcrV, HCDR3-PcrV, LCDR1-PcrV, LCDR2-PcrV, andLCDR3-PcrV, wherein HCDR1-PcrV has the amino acid sequence of SEQ ID NO:2, HCDR2-PcrV has the amino acid sequence of SEQ ID NO: 3, HCDR3-PcrVhas the amino acid sequence of SEQ ID NO: 4, LCDR1-PcrV has the aminoacid sequence of SEQ ID NO: 6, LCDR2-PcrV has the amino acid sequence ofSEQ ID NO: 7, and LCDR3-PcrV has the amino acid sequence of SEQ ID NO:8.
 2. The method of claim 1, wherein the infection is pneumonia,bacteremia, bone infection, joint infection, skin infection, burninfection, wound infection, or any combination thereof.
 3. The method ofclaim 1, wherein the infection is a nosocomial infection. 4-5.(canceled)
 6. The method of claim 1, wherein the bispecific antibodycomprises a heavy chain constant region domain-1 (CH1) comprising theamino acid sequence of SEQ ID NO:
 16. 7. The method of claim 1, whereinthe bispecific antibody comprises a first linker (L1) and a secondlinker (L2), wherein L1 and L2 are the same or different, andindependently comprise (a) [GGGGS]n, wherein n is 0, 1, 2, 3, 4, or 5(SEQ ID NO: 23), (b) [GGGG]n, wherein n is 0, 1, 2, 3, 4, or 5 (SEQ IDNO: 24), or a combination of (a) and (b).
 8. The method of claim 1,wherein the bispecific antibody comprises a first heavy chain hingeregion fragment (H1) comprising the amino acid sequence EPKSC (SEQ IDNO: 17).
 9. The method of claim 1, wherein the bispecific antibodycomprises a second heavy chain hinge region fragment (H2) comprising theamino acid sequence DKTHTCPPCP (SEQ ID NO: 18).
 10. The method of claim1, wherein the bispecific antibody comprises a heavy chain constantregion domain-2 (CH2) and a heavy chain constant region domain-3 (CH3),wherein the CH2-CH3 comprises the amino acid sequence of SEQ ID NO: 19.11. (canceled)
 12. The method of claim 1, wherein the heavy chaincomprises the amino acid sequence of SEQ ID NO: 21, and the light chaincomprises the amino acid sequence of SEQ ID NO:
 22. 13. (canceled) 14.The method of claim 1, wherein at the time of the administration thesubject is colonized with Pseudomonas aeruginosa in the respiratorytract. 15-16. (canceled)
 17. The method of claim 14, wherein thesubject's respiratory tract is colonized with P. aeruginosa one, two,three, or four days prior to administration of the bispecific antibody.18. (canceled)
 19. The method of claim 14, wherein the subject'srespiratory tract is additionally colonized by Staphylococcus aureus atthe time of administration of the bispecific antibody.
 20. The method ofclaim 1, wherein the subject is about to be hospitalized, is currentlyhospitalized, was recently hospitalized, is on a mechanical ventilator,or a combination thereof.
 21. (canceled)
 22. The method of claim 1,wherein the subject is hospitalized in an intensive care unit (ICU) andis intubated.
 23. The method of claim 20, wherein the subject ismechanically ventilated through an endotracheal or nasotracheal tube.24. The method of claim 20, wherein the administration reduces the riskof pneumonia while on mechanical ventilation.
 25. (canceled)
 26. Themethod of claim 1, wherein 1500 mg of the bispecific antibody isadministered.
 27. The method of claim 1, wherein 3000 mg of thebispecific antibody is administered.
 28. The method of claim 14, whereinthe subject has not received antibiotics active against the P.aeruginosa strain with which the subject is colonized prior toadministration of the bispecific antibody. 29-32. (canceled)
 33. Themethod of claim 1, wherein the subject maintains a serum concentrationof the bispecific antibody of at least about 1 μg/ml through day 21following administration of the bispecific antibody. 34-35. (canceled)